Varied sensitivity to therapy of HIV-1 strains in CD4+ lymphocyte sub-populations upon ART initiation

Abstract

Background: Although antiretroviral therapy (ART) has proven its success against HIV-1, the long lifespan of infected cells and viral latency prevent eradication. In this study we analyzed the sensitivity to ART of HIV-1 strains in naïve, central memory and effector memory CD4+ lymphocyte subsets.

Methods: From five patients cellular HIV-1 infection levels were quantified before and after initiation of therapy (2-5 weeks). Through sequencing the C2V3 region of the HIV-1 gp120 envelope, we studied the effect of short-term therapy on virus variants derived from naïve, central memory and effector memory CD4+ lymphocyte subsets.

Results: During short-term ART, HIV-1 infection levels declined in all lymphocyte subsets but not as much as RNA levels in serum. Virus diversity in the naïve and central memory lymphocyte populations remained unchanged, whilst diversity decreased in serum and the effector memory lymphocytes. ART differentially affected the virus populations co-circulating in one individual harboring a dual HIV-1 infection. Changes in V3 charge were found in all individuals after ART initiation with increases within the effector memory subset and decreases found in the naïve cell population.

Conclusions: During early ART virus diversity is affected mainly in the serum and effector memory cell compartments. Differential alterations in V3 charge were observed between effector memory and naïve populations. While certain cell populations can be targeted preferentially during early ART, some virus strains demonstrate varied sensitivity to therapy, as shown from studying two strains within a dual HIV-1 infected individual.

Figure 1

Viral load and cellular infection levels before and after initiation of ART. (A) Viral load values were calculated before (-) and after (+) initiation of ART and are plotted on logarithmic scale. The median decline in copy number is inserted within the graph. (B) The number of HIV-1 gag copies per 10⁵ cells of the respective cell subset is depicted on the y-axis in logarithmic scale. An occasional subset was not included due to a large difference between the duplicate measurements.

Figure 2

Neighbor-joining phylogenetic analysis of the gp120 virus sequences. The Kimura-2 parameter and 100 replicates were used to calculate nucleotide distances and sequences from the Los Alamos HIV-1 database were used as reference strains. Circles indicate sequences from serum, diamonds from naïve CD4⁺ T cells, triangle from central memory and squares from effector memory cells. (A) Phylogeny of the strains isolated before initiation of therapy (B) Phylogeny of the strains isolated after therapy initiation. The black curved lines indicate strains from the effector memory population and the white curved lines indicate strains from serum. The dotted line indicates the two virus strains co-circulating in subject M12020.

Figure 3

Diversity and divergence of the viral quasi-species. (A) From each patient pair-wise nucleotide distances before (-) and after (+) initiation of therapy were calculated for each cell subset and serum. Nucleotide distance is presented as percentage and the red bar represents the median value. Pair-wise distances between both time-points were calculated (d) and when this value was higher than the diversity of either time-point it was identified as viral divergence, indicated by an asterisk. Statistical significance was calculated for the difference in diversity before and after therapy start; *** = p < 0.0001. Data from the effector memory subset of M16394 was not available.

Figure 4

Inter-group nucleotide diversity. Before (ART-; white bars) and after (ART+; grey bars) therapy initiation, the mean difference in nucleotide distance was calculated using the Neighbor-joining model and the Kimura-2 parameter method. Each viral compartment was compared with all others (1: naïve - central memory, 2: naïve - effector memory and 3: central memory - effector memory).

Figure 5

Change in V3 charge after initiation of ART. From all cellular subsets and serum the net V3 charge of each viral clone was calculated. The net V3 charges of all patients were grouped per time-point before (-) and after (+) initiation of ART. The graph depicts the mean value with standard deviation. *** = p < 0.0001 and ns = not significant.